Effects of Arsenic on Osteoblast Differentiation in Vitro and on Bone Mineral Density and Microstructure in Rats

Background: Arsenic is a ubiquitous toxic element and is known to contaminate drinking water in many countries. Several epidemiological studies have shown that arsenic exposure augments the risk of bone disorders. However, the detailed effect and mechanism of inorganic arsenic on osteoblast differentiation of bone marrow stromal cells and bone loss still remain unclear. Objectives: We investigated the effects and mechanism of arsenic on osteoblast differentiation in vitro and evaluated bone mineral density (BMD) and bone microstructure in rats at doses relevant to human exposure from drinking water. Methods: We used a cell model of rat primary bone marrow stromal cells (BMSCs) and a rat model of long-term exposure with arsenic-contaminated drinking water, and determined bone microstructure and BMD in rats by microcomputed tomography (μCT). Results: We observed significant attenuation of osteoblast differentiation after exposure of BMSCs to arsenic trioxide (0.5 or 1 μM). After arsenic treatment during differentiation, expression of runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), and osteocalcin in BMSCs was inhibited and phosphorylation of enhanced extracellular signal-regulated kinase (ERK) was increased. These altered differentiation-related molecules could be reversed by the ERK inhibitor PD98059. Exposure of rats to arsenic trioxide (0.05 or 0.5 ppm) in drinking water for 12 weeks altered BMD and microstructure, decreased Runx2 expression, and increased ERK phosphorylation in bones. In BMSCs isolated from arsenic-treated rats, osteoblast differentiation was inhibited. Conclusions: Our results suggest that arsenic is capable of inhibiting osteoblast differentiation of BMSCs via an ERK-dependent signaling pathway and thus increasing bone loss. Citation: Wu CT, Lu TY, Chan DC, Tsai KS, Yang RS, Liu SH. 2014. Effects of arsenic on osteoblast differentiation in vitro and on bone mineral density and microstructure in rats. Environ Health Perspect 122:559–565; http://dx.doi.org/10.1289/ehp.1307832


Introduction
Environmental arsenic pollution causes a signifi cant global problem for human health. Arsenic in the environment contaminates soil and ground water and is released to food and drinking water. In certain areas of the world where arsenic contamination is endemic, such as Bangladesh, China, India, Mexico, Romania, Taiwan, and others, arsenic-related disease is prevalent as a result of drinking arsenic-contaminated water (Garelick et al. 2008). Arsenic exposure has been associated with increased incidence of various health conditions such as hyper tension, cardiovascular disorders, skin lesions, cancer, and spontaneous pregnancy loss (Abhyankar et al. 2012;Bloom et al. 2010;Chen et al. 2009). In areas with high levels of arsenic contamination in drinking water, increased mortality has been reported for males and females with several cancers, including bone cancer, compared with the local reference population (Tsai et al. 1999). Arsenic is known to replace phosphorus and localize in the bone, where it may remain for years. Feussner et al. (1979) observed bone marrow abnormality in a patient with severe arsenic poisoning. Some epidemiological studies have reported that arsenic exposure augments the risk of bone dis orders (Akbal et al. 2013;Haag et al. 1974;Lever 2002). In a recent study, Akbal et al. (2013) found that arsenic exposure in male participants was associated with bone metabolism, suggesting that arsenic exposure may be a possible cause of osteopenia. Hu et al. (2012) observed that short-term exposure of high-dose inorganic arsenic (10 mg/kg/day) to rats through an unusual route of arsenic exposure (intra peritoneal injection) affected bone remodeling. However, the mechanism of arsenic on the bone system are still unclear.
Arsenic exposure induces complex modes of action to disturb physiological functions (Abhyankar et al. 2012;Bailey et al. 2013). Arsenic stress could lead to activation of cellu lar and molecular signal transduction in target organs (Qian et al. 2003;Wang et al. 2012). Extracellular signal-regulated kinase (ERK), a member of mitogen-activated protein kinases (MAPK), has been found to contribute to arsenic-induced toxicological responses (Bonati et al. 2006;Ivanov and Hei 2013;Wang et al. 2013). ERK activation also plays an important role in osteoblast differentiation and osteo clast formation (Lai et al. 2001;Matsushita et al. 2009). In addition, ERK can regulate the expression of osteo blast differentiation-related signaling molecules, such as runt-related transcription factor 2 (Runx2), bone morpho genetic protein-2 (BMP-2), and core-binding factor a1 (Celil and Campbell 2005;Wu et al. 2012). However, the effect of arsenic on ERK signaling during osteo blast differentiation still remains unclear. In the present study, we hypothesized that low-dose inorganic arsenic disturbs osteo blast differentiation from bone marrow stromal cells (BMSCs) through an ERK signaling pathway and induces bone loss. Our results showed that low-dose inorganic arsenic significantly decreased osteo blast differentiation from BMSCs via an ERK-dependent pathway in vitro and in vivo.

Animal experiments. The Animal Research
Committee of the College of Medicine, National Taiwan University, approved and conducted the study in accordance with the guidelines for the care and use of laboratory animals. A total of 32 male Wistar rats (6-8 weeks of age) were purchased from BioLASCO (Taipei, Taiwan). Two rats were housed per standard rat micro isolator cage on aspen chip bedding in an animal room maintained at 22 ± 2°C with a 12-hr light/dark cycle. The animals were treated humanely and with regard for alleviation of suffering. Rats were provided standard chow diet (LabDiet #5053; LabDiet, St. Louis, MO, USA) and deionized, sterile water ad libitum. The maximum contaminant level of arsenic in drinking water in Taiwan is 0.01 ppm. For in vivo experiments, rats were randomly divided into three groups (8 animals/group), with each group receiving 0, 0.05, or 0.5 ppm As 2 O 3 (arsenic trioxide; Sigma-Aldrich, St. Louis, MO, USA) in drinking water for 12 weeks.
After 12 weeks of arsenic exposure, 4 animals from each exposure group were sacrificed and the left and right tibias were removed. Left tibias were fixed in Background: Arsenic is a ubiquitous toxic element and is known to contaminate drinking water in many countries. Several epidemiological studies have shown that arsenic exposure augments the risk of bone disorders. However, the detailed effect and mechanism of inorganic arsenic on osteoblast differentiation of bone marrow stromal cells and bone loss still remain unclear. oBjectives: We investigated the effects and mechanism of arsenic on osteoblast differentiation in vitro and evaluated bone mineral density (BMD) and bone micro structure in rats at doses rele vant to human exposure from drinking water. Methods: We used a cell model of rat primary bone marrow stromal cells (BMSCs) and a rat model of long-term exposure with arsenic-contaminated drinking water, and determined bone micro structure and BMD in rats by micro computed tomography (μCT). results: We observed significant attenuation of osteo blast differentiation after exposure of BMSCs to arsenic trioxide (0.5 or 1 μM). After arsenic treatment during differentiation, expression of runtrelated transcription factor-2 (Runx2), bone morpho genetic protein-2 (BMP-2), and osteocalcin in BMSCs was inhibited and phosphorylation of enhanced extracellular signal-regulated kinase (ERK) was increased. These altered differentiation-related mole cules could be reversed by the ERK inhibitor PD98059. Exposure of rats to arsenic trioxide (0.05 or 0.5 ppm) in drinking water for 12 weeks altered BMD and micro structure, decreased Runx2 expression, and increased ERK phosphorylation in bones. In BMSCs isolated from arsenic-treated rats, osteoblast differentiation was inhibited. conclusions: Our results suggest that arsenic is capable of inhibiting osteoblast differentiation of BMSCs via an ERK-dependent signaling pathway and thus increasing bone loss.  -buffered saline (PBS) containing 4% paraformaldehyde for 48 hr; BMD analysis was then performed by micro computer tomography (μCT). Right tibias were decalcified with 10% sodium EDTA solution at 4 o C for 1 month. The samples were then embedded in paraf n and sectioned to a thickness of 4 μm for immuno fluorescence staining. The tibias and femurs from the remaining 4 animals/group were used to prepare BMSCs .
Bone marrow cells. Primary BMSCs were isolated from rats and cultured with or without the differentiation medium, as previously described (Çelebi et al. 2010). Briefly, BMSCs were prepared by removing tibias and femurs from rats under anesthesia (sodium pento barbital; Sigma-Aldrich) and flushing the bone marrow cavity with growth medium (α-minimum essential medium; αMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL strepto mycin (all from Life Technologies, Carlsbad, CA, USA). Cells were then cultured in growth medium at 37 o C in a humidified atmosphere of 5% CO 2 in air. After 1 week of cell expansion, the adherent cells were treated with differentiation inducers (10 -8 M dexamethasone, 10 μM β-glycerophosphate, and 50 μg/mL ascorbic acid; all from Sigma-Aldrich) in the medium to induce osteoblast differentiation.
Alkaline phosphatase (ALP) activity assay. We examined ALP activity using an ALP activity assay kit (Alkaline Phosphatase liquicolor; Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) following the manu facturer's instructions. Briefly, BMSCs (2.5 × 10 4 /well) isolated from control or As 2 O 3 -treated rats were treated with 0, 0.5, or 1 μM As 2 O 3 , with or without 20 μM PD98059, for 7 days in differentiation medium. The medium was changed every 3 days. Cells were harvested using RIPA buffer and centrifuged at 13,000 × g for 30 min. We measured ALP activity in the supernatant; absorbance was read at 420 nm. Each sample was normalized by protein level.
Calcium measurement. To detect calcium concentrations in culture medium, we used a calcium concentration assay kit (o-cresolphthalein complexone kit; Teco Diagnostics, Anaheim, CA, USA) following the manufacturer's instructions. Briefly, BMSCs (5 × 10 5 cells/plate) isolated from control rats were cultured in differen tiation medium with 0, 0.5, or 1 μM As 2 O 3 for 5 or 14 days; the medium was changed every 3 days. Fifty micro liters of culture media collected at the end of day 5 or day 14 was mixed with working reagent (o-cresolphthalein complexone) and calcium buffer for 2-3 min at room temperature. The absorbance was detected at 570 nm and the concentration calculated by the standard curve.
Mineralized nodule formation assay. Mineralization was detected by Alizarin red S staining. Briefly, BMSCs (2.5 × 10 4 /well) isolated from control or As 2 O 3 -treated rats were cultured in differentiation medium with 0, 0.5, or 1 μM As 2 O 3 , with or without 20 μM PD98059, for 20 days. The medium was changed every 3 days. Cells were washed with PBS buffer, fixed in ice-cold 75% (vol/vol) ethanol, and then stained with 2% (wt/vol) Alizarin red S (Sigma-Aldrich). The stained cells were incubated with 10% (wt/vol) cetylpyridinium chloride (Sigma-Aldrich) to elute the Alizarin red S, and the solution was collected from the cells and measured at an absorbance at 550 nm.
μCT evaluation of trabecular and cortical bones. We assessed BMD in tibias by μCT scanning, as described previously (Takahata et al. 2012). Briefly, bones were scanned using μCT (Skyscan 1176; Bruker-MicroCT, Kontich, Belgium) with iso tropic high resolution. Tibias were scanned at 80 keV and 309 μA with an aluminum plus copper filter, and the images were collected. Quantification of trabecular and cortical bone morpho metric indices was performed in the regions of metaphysis and diaphysis in the proximal tibias, respectively. The trabecular/ cortical BMD, trabecular bone volume fraction [bone volume/total volume (BV/TV)], trabecular/cortical thickness, and cortical area were measured and analyzed by Skyscan CTAn v.1.1.7 software (Bruker-MicroCT).
Immunofluorescence staining. The 4-μm sections of paraffin-embedded tibia were deparafnized with xylene and washed with 90%, 75%, and 50% alcohol for 5 min each. Sections were then treated with 3% hydrogen peroxide-methanol solution to eliminate endogenous peroxidase activity and incubated with protease type XIV (0.5 mg/mL; Sigma-Aldrich) for 10 min. Tibia sections were blocked with 5% goat serum for 1 hr to prevent non specific binding, incubated overnight with the antibody for either Runx2 or phosphorylated ERK (1:200), and then treated with anti-rabbit or anti-mouse FITC (fluorescein isothio cyanate)-labeled secondary antibody (1:500; Sigma-Aldrich) for 1 hr. Finally, the sections were counter stained with Hoechst 33258 (1 μg/mL; Sigma-Aldrich).
Statistical analysis. Statistical analyses were performed using SPSS-16.0 software (IBM SPSS Statistics, Armonk, NY, USA). Data are expressed as mean ± SD. Data were analyzed for statistical significance using one-way analysis of variance followed by Holm-Sidak post analysis to test for differences between groups; p ≤ 0.05 was considered statistically significant. Figure 1A, BMSCs treated with As 2 O 3 at 3-15 μM for 48 hr showed decreased viability, but BMSCs treated with lower doses of As 2 O 3 (0.5 and 1 μM) for 3-18 days showed no change in cell viability ( Figure 1B). ALP was significantly decreased in BMSCs treated with 1 μM As 2 O 3 at day 5 and in those treated with 0.5 or 1 μM As 2 O 3 at day 7 (Figure 2A). We observed a decrease in calcium absorption in BMSCs treated with 0.5 or 1 μM As 2 O 3 at 14 days but not at 5 days ( Figure 2B). A decrease in osteo blast mineraliza tion occurred in BMSCs treated with 0.5 or 1 μM As 2 O 3 at days 14 and 20 ( Figure 2C). We also observed mRNA expression of the osteo blasto genic markers BMP-2 and osteocalcin Bmp2 was decreased by 0.5 or 1 μM As 2 O 3 at day 5, and osteocalcin was decreased by 1 μM As 2 O 3 at days 10 and 14 ( Figure 2D). These results suggest that nontoxic low-dose As 2 O 3 is capable of attenuating osteoblast differentiation of BMSCs.
Arsenic altered bone micro structure and osteo blast differentiation in rats. Twelve weeks after exposure to 0.05 or 0.5 ppm As 2 O 3 in drinking water, body weights of rats were not significantly affected (control, 334.3 ± 21.5; 0.05 ppm, 339.9 ± 19.2; 0.5 ppm, 345.5 ± 5.0 g; n = 8/group). In As 2 O 3 -treated rats, micro structures in trabecular and cortical bone were altered ( Figure 5A). In addition, BMD, BV/TV, and thickness of trabecular bone ( Figure 5B), and BMD, cortical area, and thickness of cortical bone ( Figure 5C) were significantly decreased. Immunofluorescence staining in bones from As 2 O 3 -treated rats displayed decreased staining for Runx2 and increased staining for phosphorylated ERK (Figure 6). In BMSCs isolated from bones of As 2 O 3 -treated rats, osteoblast differentiation ( Figure 7A) and mineraliza tion ( Figure 7B) were significantly decreased, and ALP activity also significantly decreased (fold of control: 0.82 ± 0.09 in the 0.05-ppm group and 0.71 ± 0.08 in 0.5-ppm group; n = 4/group; p < 0.05). These results suggest that arsenic exposure caused the inhibition of osteoblast differentiation and altered bone micro structure and BMD in rats.

Discussion
The main source of arsenic exposure in humans is arsenic-contaminated drinking water. Singh et al. (2007) estimated that arsenic concentrations in contaminated areas are several times higher than the maximum contamination level (the standard set by the World Health Organization and the U.S. Environmental Protection Agency) of 10 μg/L (0.01 ppm). Approximately 6 million people in West Bengal might be exposed to drinking water containing arsenic at > 50 μg/L (0.05 ppm) (Centeno et al. 2007).
In an epidemiological study in Antofagasta, Chile, Borgono and Greiber (1971) observed that arsenic-related health problems resulted from exposure to contaminated drinking water, with arsenic concentrations as high as 800 μg/L (0.8 ppm). Arsenic has also associated with an increase in liver cancer mortality in both sexes, when arsenic levels are > 0.64 mg/L (0.64 ppm) (Lin et al. 2013). As 2 O 3 has been reported to induce partial differentiation in acute pro myelocytic leukemia cells at low concentrations (0.1-0.5 μM; about 0.02-0.1 ppm) but induce apoptosis at relatively high concentrations (0.5-2 μM; about 0.1-0.4 ppm) (Chen et al. 1997). Similarly, Yen et al. (2010Yen et al. ( , 2012 reported that low-dose As 2 O 3 (0.1-0.5 μM; about 0.02-0.1 ppm) dose dependently inhibited in vitro skeletal muscle cell differentiation but higher concentrations (1-10 μM; about 0.2-2 ppm) induced apoptosis. In addition, Kesari et al. (2012) observed significant genetic damage in mice exposed to arsenic at the human equivalent reference dose (0.3 μg/kg/day), as well as its multiples (1.5-30 μg/kg/day). Obvious DNA damage was observed in bone marrow cells of mice exposed to arsenic (0.05 and 5 ppm) for 180 days (Singh et al. 2010). Exposure to 2.5-5 μM arsenite (about 0.5-1 ppm) could enhance the differentiation of preosteo clastic cells, suggesting that arsenic may result in increased bone resorption *p < 0.05, compared with the 0-μM As 2 O 3 + DM group. # p < 0.05, compared with the respective As 2 O 3 group without PD98059.   Szymczyk et al. 2006). Rats treated with arsenite (0.21 mg/kg/day) for 45 days have also been found to have increased thickness of the growth cartilage and the hypertrophic zone, as well as trabeculae sealed to the cartilage (Odstrcil Adel et al. 2010). Recently, Hu et al. (2012) observed that, in vitro, relatively high concentrations of inorganic arsenic (≥ 2 μM; about 0.4 ppm) significantly decreased the differentiation of rat calvaria pre osteoblasts; furthermore, they also found that short-term, high-dose arsenic (10 mg/kg/day for 4 weeks) administered by intra peritoneal injection, an unusual route of arsenic exposure, decreased both femur BMD and trabecular bone volume in rats. In the present study, we found that sub micromolar As 2 O 3 (0.5 and 1 μM) significantly reduced osteoblast differentiation of BMSCs in vitro.
We also found that long-term exposure of rats to As 2 O 3 in drinking water (0.05 and 0.5 ppm, 12 weeks)-doses found in human drinking water in arsenic-contaminated areas-significantly decreased BMD and bone Runx2 expression, increased bone ERK phosphorylation, and decreased osteoblast differentiation of BMSCs. These results suggest that exposure to arsenic at doses relevant to human exposure from drinking water may alter osteoblast differentiation of bone marrow cells and induce bone loss. Arsenic can exist in the environment in several valency states (-3, 0, +3, and +5). It is mostly found in inorganic form as tri valent arsenite (As 3+ ) and pentavalent arsenate (As 5+ ) in natural water. The ratio of As 3+ /As 5+ in water can greatly vary. In As-rich groundwater in Bangladesh, the ratios of As 3+ to total arsenic range from about 0.1 to 0.9, but are typically around 0.5-0.6 (Jiang et al. 2013). In a previous study, Smedley and Kinniburgh (2002) found that the kinetic of oxygenation of As 3+ is slow in the slightly acid range, around pH 5, and it is stable in the anoxic solution for up to 3 weeks. As 2 O 3 , a trivalent arsenic compound, can be released into air and water by natural or industrial processes, and it can form arsenite in alkaline solution. In the present study, to prevent or minimize oxidation of As 2 O 3 , we prepared the cell culture medium and the rats' drinking water containing As 2 O 3 every 2 and 3 days, respectively.
The ERK signaling pathway is involved in cell-matrix interactions in bone and the process of osteoblast differentiation (Ghosh-Choudhury et al. 2007;Lai et al. 2001;Wirries et al. 2013). Wu et al. (2012) suggested that osteoblastic differentiation of BMSCs is regulated by an ERK-related pathway. Exposure to arsenic has been reported to elevate ERK phosphorylation in various kinds of cells, such as endothelial cells (Wang et al. 2012), keratino cytes (Phillips et al. 2013), and neuronal mesencephalic cells (Felix et al. 2005), protecting against arsenicinduced damage. In contrast, a recent study found that sodium arsenite diminishes neuronal stem cell differentiation via over activation of an ERK signaling pathway (Ivanov and Hei 2013). Activation of ERK signaling has also been shown to be involved in the inhibition of osteo blastic differentiation of vascular smooth muscle cells by ghrelin (Liang et al. 2012) or taurine (Liao et al. 2008). Tang et al. (2008) reported that PD98059 (20 μM) did not decrease ALP activity in rat osteoblasts. Lin et al. (2011) found that 20 μM PD98059 potentially induced rat pre osteoblast differentiation, whereas Bai et al. (2013) reported that 10 μM PD98059 decreased osteoblast differentiation in rabbit BMSCs. In the present study, we found that As 2 O 3 activated ERK activation during osteoblast differentiation of BMSCs and that PD98059 significantly reversed As 2 O 3 -inhibited osteoblast differentiation, suggesting that arsenic may inhibit osteoblasto genesis through an ERKdependent signaling pathway. Taken together, these findings (Bai et al. 2013;Lin et al. 2011;Tang et al. 2008) and our results suggest that ERK activation can lead either to stimulation or inhibition of osteoblast differentiation pathways, depending on the system. Runx2 is a master transcription factor that regulates bone formation and subsequently forms the fully functional osteoblasts (Lee et al. 2000). Celil and Campbell (2005) found that Runx2 activation is regulated by an ERK-dependent signaling pathway in human mesen chymal stem cells. Moreover, nuclear factor E2 p45-related factor 2 (Nrf2), a transcription factor for the regulation of many detoxifying and anti oxidative genes, is known to be activated by ERK signaling (Cai et al. 2012;Khan et al. 2011). Hinoi et al. (2006) suggested that Nrf2 can negatively regulate osteoblast differentiation via an inhibition of the Runx2-dependent transcriptional activity (Hinoi et al. 2006). In the present study, we found that As 2 O 3 activated ERK phosphorylation and inhibited Runx2 expression during osteoblast differentia tion, which could be reversed by ERK inhibitor. The immunofluorescence co-localization of Runx2 and phosphorylated ERK has been shown in osteoblast cells (Li et al. 2010). The immunofluorescence staining for Runx2 and phosphorylated ERK in bones ( Figure 6) might be mainly localized in osteoblast cells. This arsenic-activated ERK that down-regulated Runx2 expression during osteoblast differentiation of BMSCs may be through an ERKactivated Nrf2 signaling pathway. However, Figure 7. Osteoblast differentiation of BMSCs isolated from bones of rats administered 0, 0.05, or 0.5 ppm As 2 O 3 in drinking water for 12 weeks. BMSCs were isolated from bones and cultured in differentiation medium for 20 days. (A) Photomicromicrographs and (B) quantitation of osteoblast mineralization. Data are mean ± SD (n = 4/group). *p < 0.05, compared with the the 0-ppm As 2 O 3 group.

Quantification of mineralization (%)
* * the role of Nrf2 in arsenic-inhibited osteoblast differentiation of BMSCs still needs to be clarified.

Conclusions
In this study, we found that low-dose arsenic significantly reduced osteoblast differentiation of bone marrow cells in vitro. In rats, long-term exposure to arsenic in drinking water-at doses relevant to human exposure from drinking water-significantly altered bone micro structure and BMD. In addition, the up-regulation of ERK and the inhibitory effect of ERK inhibitor indicated that arsenic inhibited osteo blasto genesis through an ERKdependent signaling pathway. Taken together, these in vitro and in vivo findings suggest that inorganic arsenic may be an environ mental risk factor for osteoporosis.